ariceca 2+ binding protein is required for tapetum · ca2+ signal is sensed by a series of ca2+...

A Rice Ca2+ Binding Protein Is Required for TapetumFunction and Pollen Formation1[OPEN]

Jing Yu, Zhaolu Meng, Wanqi Liang, Smrutisanjita Behera, Jörg Kudla, Matthew R. Tucker, Zhijing Luo,Mingjiao Chen, Dawei Xu, Guochao Zhao, Jie Wang, Siyi Zhang, Yu-Jin Kim, and Dabing Zhang*

Joint International Research Laboratory of Metabolic & Developmental Sciences, Shanghai Jiao TongUniversity-University of Adelaide Joint Centre for Agriculture and Health, School of Life Sciences andBiotechnology, Shanghai Jiao Tong University, Shanghai 200240, China (J.Y., Z.M., W.L., Z.L., M.C., D.X., G.Z.,J.W., S.Z., Y.-J.K., D.Z.); Institut für Biologie und Biotechnologie der Pflanzen, Westfälische Wilhelms-Universität Münster, Schlossplatz 7, 48149 Münster, Germany (J.K.); Department of Oriental MedicinalBiotechnology, College of Life Science, Kyung Hee University, Yongin 446-701, Republic of Korea (Y.-J.K);Indian Institute of Chemical Biology, Council of Scientific and Industrial Research, 700 032 West Bengal, India(S.B.); and School of Agriculture, Food and Wine, University of Adelaide, Waite Campus, Urrbrae, SA 5064,Australia (M.R.T., D.Z.)

ORCID IDs: 0000-0002-9938-5793 (W.L.); 0000-0003-4661-6700 (M.R.T.); 0000-0002-9079-5980 (M.C.); 0000-0001-8324-450X (D.X.);0000-0002-2825-9554 (G.Z.); 0000-0003-2562-615X (Y.-J.K.); 0000-0002-1764-2929 (D.Z.).

In flowering plants, successful male reproduction requires the sophisticated interaction between somatic anther wall layers andreproductive cells. Timely degradation of the innermost tissue of the anther wall layer, the tapetal layer, is critical for pollendevelopment. Ca2+ is a well-known stimulus for plant development, but whether it plays a role in affecting male reproductionremains elusive. Here we report a role of Defective in Exine Formation 1 (OsDEX1) in rice (Oryza sativa), a Ca2+ binding protein,in regulating rice tapetal cell degradation and pollen formation. In osdex1 anthers, tapetal cell degeneration is delayed anddegradation of the callose wall surrounding the microspores is compromised, leading to aborted pollen formation and completemale sterility. OsDEX1 is expressed in tapetal cells and microspores during early anther development. Recombinant OsDEX1 isable to bind Ca2+ and regulate Ca2+ homeostasis in vitro, and osdex1 exhibited disturbed Ca2+ homeostasis in tapetal cells.Phylogenetic analysis suggested that OsDEX1 may have a conserved function in binding Ca2+ in flowering plants, and geneticcomplementation of pollen wall defects of an Arabidopsis (Arabidopsis thaliana) dex1 mutant confirmed its evolutionaryconservation in pollen development. Collectively, these findings suggest that OsDEX1 plays a fundamental role in thedevelopment of tapetal cells and pollen formation, possibly via modulating the Ca2+ homeostasis during pollen development.

Higher plants alternate their life cycle between sporo-phytic and gametophytic generations that result from twosequential processes: sporogenesis and gametogenesis(Goldberg et al., 1993). Formation of the male gameto-phyte is a complex process that starts with anther mor-phogenesis, followed by microspore formation viameiosis and mitosis (Ma, 2005; Gómez et al., 2015; Zhangand Liang, 2016). The somatic cell layers surrounding themicrosporocytes include the epidermis, endothecium,middle layer, and tapetum,which are required for normalpollen development. The differentiation of the innermosttapetal cell layer is crucial for pollen formation (KelliherandWalbot, 2012; Fu et al., 2014; Zhang and Yang, 2014).After the formation of tapetal cells, subsequent tapetal celland callose degradation, as well as primexine formation,are vital for pollen development (Ma, 2005; Li et al., 2006;Wu and Cheun, 2000; Paxson-Sowders et al., 2001;Ariizumi et al., 2004; Li et al., 2011a; Chang et al., 2012; Jiet al., 2013; Niu et al., 2013; Sun et al., 2013).

During tapetum development, regulated tapetal celldeath is of vital importance for primexine formation,sporopollenin synthesis, and exine formation (Shi et al.,

1 This research was supported by the National Key TechnologiesResearch and Development Program of China, Ministry of Scienceand Technology (grant no. 2016YFD 0100804); the National NaturalScience Foundation of China (grants no. 31322040 and no. 31271698);the National Key Basic Research Developments Program of the Min-istry of Science and Technology, China (grant no. 2013 CB126902); theInnovative Research Team, Ministry of Education; the 111 Project(grant no. B14016); the Science and Technology Commission ofShanghai Municipality (grant no. 13JC1408200); and the NationalTransgenic Major Program (grant no. 2016ZX08009003-003-007).

* Address correspondence to [email protected] author responsible for the distribution of materials integral to

the findings presented in this article in accordance with the policydescribed in the Instructions for Authors (www.plantphysiol.org) is:Dabing Zhang ([email protected]).

J.Y. performed most of the experiments; Z.M. conceived the originalscreening of mutant identification; S.B. and J.K. analyzed the Ca2+ con-centration; J.K. analyzed the Ca2+ binding activity; M.R.T. performed thecallose immunolabeling; Z.L. andM.C. generated F2 population for map-ping; D.X., G.Z., J.W., and S.Z. performed map-based cloning; Y.J.K. con-tributed for preparation of the manuscript; D.Z. and W.L. supervised theproject; D.Z. complemented the writing.

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1772 Plant Physiology�, November 2016, Vol. 172, pp. 1772–1786, www.plantphysiol.org � 2016 American Society of Plant Biologists. All Rights Reserved.

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2015; Zhang and Liang 2016). Until now, several tran-scription factors and their associated targets have beenreported to play role in tapetal cell death (Sorensenet al., 2003; Jung et al., 2005; Li et al., 2006; Aya et al.,2009; Xu et al., 2010; Li et al., 2011a; Niu et al., 2013;Ji et al., 2013; Fu et al., 2014; Ko et al., 2014). In rice(Oryza sativa), mutations in the basic helix-loop-helix(bHLH) transcription factor TDR Interacting Protein2(TIP2), also called bHLH142, show compromised inneranther wall layer differentiation and defects in micro-spore development (Fu et al., 2014; Ko et al., 2014). Amutant of the bHLH transcription factor, UndevelopedTapetum1(UDT1), displays abnormal developmentand degeneration of the tapetum and middle layer(Jung et al., 2005). gamyb, which encodes a MYB tran-scription factor, shows aborted tapetum degradation(Aya et al., 2009). Another bHLH transcription factor,Eternal Tapetum1 (EAT1), also called DTD, as wellas one PHD finger protein, Persistent Tapetal Cell1(PTC1), regulate microspore development via control-ling tapetal cell death (Li et al., 2011a; Ji et al., 2013; Niuet al., 2013). TDR and EAT1 directly regulate the ex-pression of proteases, which are regarded as executorsfor programmed cell death (PCD) in animals (Li et al.,2006; Niu et al., 2013; Woltering, 2010). For instance,EAT1 regulates the two aspartic proteases OsAP25 andOsAP37, which trigger PCD in both yeast and plants(Niu et al., 2013). As a positive tapetal PCD determi-nant, TDR regulates the expression of OsCP1, a Cysprotease that is involved in tapetal degradation (Leeet al., 2004; Li et al., 2006; Niu et al., 2013). In addition,OsCP1 is also regulated by two ATP-dependent RNAhelicases, AIP1 and AIP2 (Li et al., 2011b).Tapetal cell death is also associated with the

degradation of callose and the formation of primex-ine on the surface of the microspore, which is the firststep of the pollen cell wall formation (Ariizumi andToriyama, 2011; Shi et al., 2015). Mutants defective incallose degradation in rice also have defects in exineformation and pollen development (Wan et al., 2011).In Arabidopsis (Arabidopsis thaliana), Defective in ExineFormation1 (DEX1), No Exine Formation1 (NEF1),Ruptured Pollen Grain1 (RPG1), Ruptured PollenGrain2 (RPG2), and No Primexine and PlasmaMembrane Undulation (NPU) are required for pri-mexine formation, and their corresponding mutantsare defective in exine formation (Paxson-Sowderset al., 2001; Ariizumi et al., 2004; Chang et al., 2012;Sun et al., 2013). After the formation of the primexine,sporopollenin was synthesized in the tapetumand transported to the microspore. Several lipidmetabolism-related genes, such as Defective PollenWall (DPW), CYP704B2, and CYP703A3 have beenreported to be essential for rice exine formation (Shiet al., 2011; Li et al., 2010; Yang et al., 2014b; Shi et al.,2015; Zhang et al., 2016). Additionally, sporopolleninprecursor transport-related genes, such as PostmeioticDeficient Anther1 (OsABCG15/PDA1), OsABCG26,and OsC6 (Zhang et al., 2010; Qin et al., 2013;Zhu et al., 2013; Zhang and Li 2014; Zhao et al.,

2015), are also required for pollen exine formation(Supplemental Fig. S1).

Up to now, the progress made toward understandingtapetal cell death is mainly restricted to the level oftranscription factors and their related regulatory effects(Sorensen et al., 2003; Li et al., 2006; Aya et al., 2009; Xuet al., 2010; Li et al., 2011a; Niu et al., 2013; Ji et al., 2013;Fu et al., 2014; Ko et al., 2014). Consequently, furtherapproaches are needed to reveal additional details oftapetal cell death regulation. Ca2+ is an essential com-ponent regulating a wide range of biological processesincluding cell division, differentiation, motility, andPCD (Poovaiah and Reddy, 1993; Trewavas andMalhó,1998; Zielinski, 1998; Reddy, 2001; Lopez-Fernandezet al., 2015). Ca2+ affects cell death, including apoptosis,autophagy, and autolysis (Groover and Jones, 1999;Giorgi et al., 2008) by triggering the increase of cytosolicCa2+([Ca2+]cyt; McConkey and Orrenius, 1997; Ferrariet al., 2002; Orrenius et al., 2003; Smaili et al., 2003;Giorgi et al., 2008). The endoplasmic reticulum (ER) isconsidered as an important Ca2+-rich organelle, andER-localized proteins have been reported to influenceCa2+ homeostasis in cells (Lam et al., 1994; Foyouzi-Youssefi et al., 2000; Kowaltowski et al., 2000; Pintonet al., 2000; Vanden Abeele et al., 2002). In Arabi-dopsis, BCL2-Associated X protein inhibitor1 acts asan inducer of ER stress-mediated PCD, which mayplay a role in affecting Ca2+ homeostasis on ER stress-mediated PCD in plants (Watanabe and Lam, 2008).Overexpression of ER-localized Bcl-2 protein pro-moted Ca2+ leakage from the ER and suppressed Ca2+

reuptake by the ER, causing the consequent increaseof [Ca2+]cyt and subsequent apoptosis (Hofer et al.,1998; Foyouzi-Youssefi et al., 2000; Vanden Abeeleet al., 2002; Ferrari et al., 2002).

Ca2+ signal is sensed by a series of Ca2+ binding pro-teins, calmodulin (CaM), calcineurin B-like proteins, andCa2+-dependent protein kinases, and activates a numberof transcriptional regulators by a kinase cascade or bydirect interaction of CaM in a Ca2+-dependent manner(Hiraga et al., 1993; Corneliussen et al., 1994; Enslen et al.,1995; Tokumitsu et al., 1995; Shi et al., 1999). Many Ca2+

sensors contain domain E and F (EF)-hand domains forCa2+ binding (Snedden and Fromm, 1998). The typicalEF-hand is a helix-loop-helix structure, in which the resi-dues with +X*+Y*+Z*-Y*-X**-Z are associated with Ca2+

binding (Day et al., 2002; Derbyshire et al., 2007; Rigdenet al., 2011). In addition, other motifs such as helix-loop-strand, helix-loop-turn, strand-loop-helix, strand-loop-strand, and several structural contexts without regularsecondary structure elements either before or after theDxDxDG-containing loop canprovide affinity for bindingCa2+ (Rigden and Galperin, 2004).

In this work, we characterized a Ca2+ binding pro-tein, OsDEX1, which is required for tapetal functionand pollen development in rice. OsDEX1 is conservedin flowering plants, and it is able to complement themutant of its Arabidopsis counterpart. These findingsprovides the first evidence for an essential role of theCa2+ homeostasis in plant pollen development.

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The Function of OsDEX1 in Rice Pollen Formation


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RESULTS

Phenotypic Analysis of osdex1

To identify genes that are required for rice male re-production, we isolated the defective in exine formation1in rice (osdex1) mutant (see below) from a rice mutantlibrary generated in ssp. japonica cv 9522 backgroundby 60Co g-ray irradiation (Chen et al., 2006), whichshowed a complete male sterility phenotype. Com-pared with wild-type plants, osdex1 mutant displayedmorphologically normal vegetative and nonreproduc-tive floral organs, but smaller and pale yellow antherslacking normal mature pollen grain, leading to com-plete male sterility (Fig. 1). All of the F1 progeny be-tween wild type and osdex1 were fertile, and thesegregation rate of F2 generation was approximately1:3 (sterility/fertility = 24:81), suggesting osdex1 incaused by a single recessive mutation.

OsDEX1 Affects Tapetal Cell Death andPrimexine Formation

Transverse sectioning was used to investigate thecellular morphological alterations of osdex1 duringpollen development, which was delineated based on aprevious report (Zhang et al., 2011). No detectablemorphological defect was observed in osdex1 anthersuntil stage 8b when the microspore mother cells of bothwild type and osdex1 had undergone the secondmeiosisto produce ellipsoidal-shaped dyads, and tapetal cellswere darkly stained with a vacuolated shape (Fig. 2, Aand E). At stage 9, the wild-type microspores had sep-arated from the tetrads and were distributed in antherlobes (Fig. 2B), while osdex1 microspores were notseparated (Fig. 2F). At stage 10, the wild-type micro-spores were covered in a thick layer of exine, displayeda round shape due to vacuolization, and the tapetumwere degenerated into a thin layer (Fig. 2C). In contrast,osdex1microspores were smaller, lacked a clear layer ofexine comparedwith the wild type, and the tapetal cellswere not degenerated but appeared expanded in some

regions (Fig. 2G). At stage 11, the wild-type tapetal cellswere completely degraded and sickle-shape micro-spores exhibited storage starch accumulation (Fig. 2D).However, osdex1 displayed a persistent tapetal layer,and the microspores showed a similar morphology toprevious stages (Fig. 2H). These observations indicatethat osdex1 exhibits delayed tapetal degradation andaborted exine formation.

Transmission electron microscopy (TEM) was usedto further investigate the defects in osdex1 pollen for-mation and tapetum degeneration. Consistent with theobservations by light microscopy, no visible morpho-logical differences were observed between the wildtype and osdex1 (Supplemental Fig. S2) until stage 8a.At stage 8b, wild-type tapetal cells became largelyvacuolated with a thin layer of cell wall (Fig. 3, A andE), and the plasma membrane (PM) of wild-type mi-crospore showed regular undulations and primexinedeposition (Fig. 3E). In osdex1 tapetal cells, where vac-uoles were reabsorbed, their cytoplasm appeared con-densed with a large number of mitochondria and theydisplayed a thicker cell wall (Fig. 3, I, M, and Q). PMundulation was not observed in mutant microsporesand primexine deposition did not occur (Fig. 3Q). Atstage 9, wild-type tapetal cells displayed awaved shapeaccompanied by an enrichment of organelles, such asmitochondria and ER, and small bubbles that likelyrepresented the early stages of orbicule development(Fig. 3, B and F). Correspondingly, a thin layer of exine(including probacula and nexine) was present on thewild-type microspore surface (Fig. 3F). Consistent withthis observation, sporopollenin precursor synthesisand transport-related genes such as DPW, CYP703A3,CYP704B2, OsC6, and PDA were highly expressed inwild-type anthers (Supplemental Fig. S3; Shi et al., 2011;Yang et al., 2014b; Li et al., 2010; Zhu et al., 2013; Zhanget al., 2010). By contrast, in osdex1, at the same stage,tapetal cells were flat with a thicker cell wall and con-tained a larger number of vacuoles with variable sizecompared to wild type (Fig. 3, J and R). Notably, thevacuoles in the mutant tapetal cells were of irregular

Figure 1. Phenotypic comparison betweenthe wild type and the osdex1 mutant.A, Wild-type plant (left) and the osdex1mutant plant (right) after heading. B, Part ofthe wild-type panicle showing the dehiscedanther (left) and part of the osdex1 panicle(right) showing a smaller anther at thepollination stage. C, Wild-type (left) andosdex1 (right) flower organs after removal ofthe palea and lemma. D, Wild-type (left)and osdex1 (right) flowers before anthesis.E, Wild-type stained pollen at stage 13.Bars = 10 cm (A), 2 cm (B), 5 mm (C and D),and 100 mm (E).

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shape and were attached to each other exhibitingmembrane ablation (Fig. 3N). Furthermore, the struc-tures of nucleus and ER seemed disrupted in the mu-tant tapetal cells (Fig. 3, O and P). No intact exinestructure was formed on the osdex1microspore surface,which only showed fragmented primexine deposition(Fig. 3R). Furthermore, no obvious expression of DPW,

CYP703A3, CYP704B2,OsC6, and PDAwas detected inthe mutant, suggesting the defective synthesis andtransport of sporopollenin precursors (SupplementalFig. S3). At stage 10, wild-type tapetal cells were thinand contained a large number of mature orbicules onthe inner surface (Fig. 3, C andG). As a result, wild-typemicrospores possessed a thick layer of exine on the

Figure 2. Bright field microscopy of transverse sec-tions showing anther and microspore development inwild type and osdex1. Locules from the anther sectionof the wild type (A–D) and osdex1 (E–H) from stage8 to stage 11. dT, degenerated tapetal layer; E, Epi-dermis; En, endothecium; M, middle layer; Msp, mi-crospores; T, tapetal layer; Tds, tetrads. Bars = 15 mm.A and E, Stage 8b. B and F, Stage 9. C andG, Stage 10.D and I, Early stage 11.

Figure 3. TEM images of the anthers from the wildtype and osdex1. A to D, TEM observation showingtapetal cells of wild-type anthers at stage 8b (A), stage9 (B), stage 10 (C), and stage11 (D). E to H, Wild-typetapetal cell wall (left) and microspore cell wall (right)at stage 8b (E), stage 9 (F), stage 10 (G), and stage11(H). I to L, TEM observation showing tapetal cells ofosdex1 anthers at stage 8b (I), stage 9 (J), stage 10 (K),and stage 11 (L). M, Higher magnification of thehighlighted region in (I) showing details in tapetalcells. N, Vacuoles fusion in osdex1 tapetal cells atstage 9. Black arrows show the attachment of vacu-oles. O and P, Higher magnification of the highlightedregion in (J) showing details in tapetal cells. Whitearrows show the breakage of vacuoles. Black arrowshows the breakage of ER. Q to T, osdex1 tapetal cellwall (left) and microspore cell wall (right) at stage 8b(Q), stage 9 (R), stage 10 (S), and stage 11 (T). Ba,Bacula; CW, cell wall; eOr, early stage of orbicules;Pb, probacula; M, mitochondria; N, nucleus; Ne,nexine; Or, orbicules; T, tapetal cells; Te, tectum;V, vacuoles. Bars =2mm(A–Cand I–K), 1mm(DandN),10 mm (L), and 0.5 mm (E–H, M, O–P, and Q–T).







surface (Fig. 3G). By contrast, osdex1 tapetal cells wereexpended in several regions because of large vacuoles(Fig. 3K). The persistent tapetal cells were covered by athick cell wall without any orbicules (Fig. 3S). More-over, mutant microspores were covered by a thin layerof darkly stained material, lacking the normal structureof exine (Fig. 3S). At stage 11, the tapetal layer in wild-type anthers was almost completely degenerated andorbicules were present around microspores forming athick exine (Fig. 3, D and H), while osdex1 exhibitedexpanded tapetal cells with large vacuoles, and ex-tremely thick cell walls (Fig. 3, L and T) withoutsculptured exine on the microspores (Fig. 3T). Theseresults suggest that OsDEX1 affects the normal tapetalcell death, synthesis of sporopollenin precursors, andthe formation of pollen wall ranging from the primex-ine to multiple-layer exine during male development.

The persistence of tapetal cells in osdex1mutants wasinvestigated using the terminal deoxynucleotidyltransferase-mediated dUTP nick-end labeling (TUNEL)assay inwhich the fluorescein-12-dUTP-labeled DNA iscatalytically incorporated into fragmented DNA, andcan be visualized by confocal laser scanning micro-scope (Li et al., 2006). The wild-type tapetum displayedDNA fragmentation from stage 8b until stage 9, andthere was no DNA fragmentation at stage 7 and stage8a. In contrast, the DNA fragmentation in osdex1 tapetalcells could be observed starting from stage 7 until stage8a, and declined at stage 8b and stage 9 (Fig. 4).Therefore, DNA fragmentation appears to be activatedearly in osdex1 tapetal cells and disrupted at the stageswhenwild-type tapetal cells showDNA fragmentation.To further assess tapetal cell death, the expressionpattern of tapetal cell death-related genes was deter-mined. OsAP25, OsAP37 and OsCP1 were not inducedin osdex1 anthers compared to wild-type anthersundergoing cell death (Supplemental Fig. S4), sug-gesting that the normal protein degradation pathwaywas disrupted in the osdex1 mutant during tapetumdevelopment.

Our observations using TUNEL and TEM sug-gest abnormal tapetal cell degeneration in osdex1.Consistent with the earlier induction of DNA frag-mentation in osdex1 tapetal cells, in osdex1we observed

up-regulation of five endonuclease-encoding genes(LOC_07g45100, LOC_01g03740, LOC_04g58850,LOC_02g50040, and LOC_08g29700) that are down-regulated in the tapetal cell death-deficient mutantsgamyb and tip2 at stage 8 (Aya et al., 2009; Fu et al.,2014). Similarly at stage 7, the genes were eitherup-regulated or showed the opposite expression pat-tern compared to gamyb and tip2. These findings are inagreement with the opposite cell death phenotype. Atstage 9 when the DNA fragmentation signal was de-clined, the expression of these genes was down-regulated(Supplemental Fig. S4).

OsDEX1 Affects Callose Degradation

The TEM analysis indicated that osdex1 microsporeswere covered in an electro-dense matrix at later stages(Fig. 5, A to D), likely indicative of abnormal callosedegradation. To further characterize the role of OsDEX1in callose dynamics during microspore development,aniline blue staining and immunolabling with a calloseantibody were performed (Figs. 5, E to L; S5, E to L). Atstage 7, both wild-type and osdex1 tetrads were labeledwith aniline blue, indicating the relatively normalsynthesis of callose in osdex1 (Supplemental Fig. S5, Eand I). A similar pattern was observed using calloseimmunolabeling, although in this case staining wasweaker around osdex1 tetrads than in wild type at stage8b (Fig. 5, F and J). This is supported by the TEM ob-servations showing a looser electron-dense matrixaround osdex1 tetrads (Supplemental Fig. S5, A and B).Also at stage 8b, both aniline blue and callose antibodystained cell-wall material in the center and the borderregion surrounding the wild-type tetrads (Figs. 5, F andJ; S5, F and J). Remarkably, in osdex1, aniline bluestaining was restricted mainly to the inner tetrad walls,suggesting some changes in cell-wall composition ofthe outer walls, possibly because of precocious yet in-complete callose degradation (Supplemental Fig. S5F).Specific callose labeling was detected by aniline blueand callose antibody directly adjoining osdex1 micro-spores, and weakly in the diffuse halos surroundingthem, until stage 10 (Figs. 5, K and L; S5, K and L) when

Figure 4. DNA fragmentation is initiated earlier andsubsequently blocked in osdex1mutant. A to H, DNAfragment signal at stage 7, stage 8a, stage 8b, andstage 9 in wild type (A–D) and osdex1 mutant (E–H).The red fluorescence shows the propidium iodidestaining of anther cells using confocal laser scanningmicroscope; the yellow fluorescence shows theTUNEL-positive nuclei staining in confocal laserscanning microscope overlays of fluorescein stainingand propidium iodide staining. Bars = 15 mm.


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staining was absent around the wild-type microspores.These results suggest that OsDEX1 affects callose deg-radation during microspore development.

Cloning and Expression Analysis of OsDEX1

To identify the gene responsible for the osdex1 phe-notype, we employed a map-based cloning approach.OsDEX1 was mapped to chromosome 3 between themarkers of Os315 and Os315-2, which incorporated a26 kb DNA fragment and seven putative genes(Supplemental Fig. S6A). After sequencing anther-expressed candidate genes within this region, a 1 bpdeletion was identified in the coding region of a genecorresponding to LOC_Os03g61050 (http://www.gramene.org/, also annotated as Os03g0825700 byhttp://rapdb.dna.affrc.go.jp/), which deletion resultedin a frame shift and subsequently an earlier termi-nation of the translation of this gene (Supplemental Fig.S6B). Moreover, three additional alleles of OsDEX1were identified, and we re-named osdex1 as osdex1-1,and the additional three alleles were designated asosdex1-2, osdex1-3, and osdex1-4 (Supplemental Fig.S6B). osdex1-2 has a one-nucleotide G to A substitutionat an intron boundary (position 4296), causing the al-ternative splicing of OsDEX1. osdex1-3 had a 374 bpdeletion in the 10th exon leading to a 286 bp deletion ofthe mRNA. osdex1-4 had a 4 bp deletion in the sixthexon leading to an earlier termination of OsDEX1. All of

the mutants exhibited similar male sterile phenotypeand expanded tapetal cells (Supplemental Fig S7). Al-lelic tests confirmed that the four mutants were indeedalleles of a single locus. The OsDEX1 transcript is3241 bp in length and contains a 2556 bp coding se-quence with 12 introns, a 180 bp 59 untranslated region,and a 505 bp 39 untranslated region (Supplemental Fig.S6B). The predicted OsDEX1 protein encompasses851 amino acids and harbors one putative N-terminalsignal peptide (amino acids 1 to 23), one integrin a-N-terminal domain (amino acids 69 to 582) containing twophenylalanyl-glycyl-glycyl-alanyl-prolyl (FG-GAP)domains (amino acids 460 to 486 and 554 to 579;PF01839; http://pfam.xfam.org), and one transmem-brane domain (amino acids 816 to 826; SupplementalFig. S6C). The integrin a-N-terminal domain is pre-dicted to mediate cell adhesion, and the FG-GAP do-main has been shown to be important for ligandbinding (Loftus et al., 1994).

To gain insights into the function of OsDEX1, quan-titative RT-PCR (qRT-PCR) analysis was used to furtherinvestigate the spatio-temporal expression pattern ofOsDEX1. In the wild type, OsDEX1 expression wasdetectable in roots, shoots, and leaves, with a preferableexpression in the anther, starting from stage 6, reachingan expressionmaximum at stage 8 and stage 9.OsDEX1expression showed a dramatic reduction in osdex1-1,particularly in anthers (Supplemental Fig. S6D). Al-though the OsDEX1 expression was high in wild-typeleaves and much reduced in the mutant, no visible

Figure 5. Callose degradation is retarded in osdex1.A to D, TEM of extracellular materials from the wildtype and osdex1 at stage 9 (A and B), and stage 10(C and D). Black arrows show the site of callose de-position. E to L, Immunolabeling of wild-type (E–H)and osdex1 (I–L) anther sections from stage 7 to stage10 observed by epifluorescence microscopy. M to P,Negative controls of immunolabeling. In (E) to (P), thegreen channel shows immunostaining with calloseantibody; blue counterstaining shows 1,4- and1,3;1,4-glucan polymers stained with 0.01% calco-fluor white; and red staining shows background auto-fluorescence. Bars = 2mm (A), 5mm (B–D), and 15mm(E–P).





http://www.gramene.org/

http://www.gramene.org/

http://rapdb.dna.affrc.go.jp/








http://pfam.xfam.org





phenotypic change was observed in mutant leavesunder normal growth conditions, suggesting no obvi-ous role of OsDEX1 or redundant gene function duringleaf development. Furthermore, in situ hybridizationalso showed thatOsDEX1was expressed in tapetal cellsand microspores from stage 8a to stage 9, and low levelat stage 10 (Supplemental Fig. S6, E to L).

OsDEX1 Has a Conserved Function in Pollen Development

To elucidate the evolutionary conservation and dis-tribution of OsDEX1, the full-length OsDEX1 proteinwas used as the query to search for its closest relativesin public databases, including the National Center forBiotechnology Information, The Arabidopsis Informa-tion Resource, Phytozome (http://www.phytozome.net/), and PLAZA (http://bioinformatics.psb.ugent.be/plaza/), which yielded 25 sequences from 24 dif-ferent species, and all of which were then included inthe phylogenetic analysis (Fig. 6). Among the 24 spe-cies, besides rice, there are monocots such as Oryzabrachyantha, dicots such as Arabidopsis, basal angio-sperm such as Amborella trichopoda, Pteridophyta suchas Selaginella moellendorffii, Bryophyta such as Phys-comitrella patens, Chlorophyta such as Chlamydomonasreinhardtii, Protozoa such as Phytomonas sp. isolate Hart,Mollusca such as Strongylocentrotus purpuratus, andProkaryotes such as Firmicutes bacterium. These data

suggested that OsDEX1 belongs to an ancient proteinsubfamily, present in both prokaryotes and eukaryotes,which was designated as DEX1 (PTHR21419:SF23) bythe panther database (http://www.pantherdb.org/).Notably, proteins in this family only exist in lower an-imals, while they are present in both higher plants andlower plants. OsDEX1 clustered together with othermonocot DEX1 sequences in the same clade, whileproteins from dicots, including the Arabidopsis ho-molog DEX1, clustered in a separate clade. In addition,the homologous eukaryotic sequences all contained atransmembrane domain, suggesting their similarmembrane localization in the cell. The FG-GAP domainwas only detectable in the sequences of floweringplants, which has been shown to be important for lig-and binding (Loftus et al., 1994). These results suggestthat OsDEX1 might have an evolutionarily conservedconserved ancient function in male gametophytedevelopment that, however, may have obtained theability ligand binding during the formation of angio-sperms.

Based on the phylogenetic analysis, OsDEX1 wasidentified as a putative ortholog of Arabidopsis DEX1.dex1 was also reported as a male sterile line due to itsdelayed primexine formation, and aborted pollen walldevelopment, but lacked characterization of its tapetaland biochemical function (Paxson-Sowders et al., 2001).The amino acid sequence of OsDEX1 was overall 66.5%

Figure 6. Phylogenetic analysis of OsDEX1 and its related proteins. Neighboring-joint analysis was performed using MEGA 6.1(see “Materials andMethods”) based on the alignment given in Supplemental Data Set S1 online of OsDEX1with themost similarOsDEX1 sequences from the species shown. The species were classified by evolutionary relationship.


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http://www.phytozome.net/

http://www.phytozome.net/

http://bioinformatics.psb.ugent.be/plaza/

http://bioinformatics.psb.ugent.be/plaza/

http://www.pantherdb.org/



identical to that of DEX1 (Supplemental Fig. S8), sug-gesting that they may have similar function. To test thishypothesis, OsDEX1 cDNA driven by DEX1 promoter(1.7 Kb) was introduced into dex1 heterozygous plants.More than 100 T1 transformants were identified,and among the dex homozygous lines containing thetransgene, 42% displayed full fertility and 25% showedpartial fertility. Notably, Alexander staining showedthat in the fully fertile lines, all the pollen grains in theanther were stained red, indicating that these pollengrains are viable (Fig. 7C). In the partially fertile lines,some anthers showed all pollen grains stained in red,while others showed a reduced amount of red-stainedpollen grains (Fig. 7D). Altogether, these results dem-onstrate that OsDEX1 fulfills a conserved function inpollen development.

OsDEX1 Is a Calcium Binding Protein

The phylogenetic analysis suggested the impor-tance of the FG-GAP domain in flowering plants, andFG-GAP domain has also been also reported to mediatecalcium binding (Tuckwell et al., 1992; Loftus et al.,1994; Springer, 1997). To investigate whether the con-served FG-GAP domain of OsDEX1 can indeed bind toCa2+, a three-dimensional (3D) structure prediction ofOsDEX1 was conducted using SWISSMODEL (http://www.swissmodel.expasy.org/), which revealed thatthe 3D structure of OsDEX1 was rich in b-sheet linked byloops (Supplemental Fig. S9), implying its potential abilityto bind Ca2+. Structural analysis showed the oxygenatoms from the side chains of the first, third, and fifthresidues in the EF-hand motif that may form a pocketsuitable for calcium atom binding (Fig. 8, A and B).An in vitro Ca2+ binding assay (Gregersen et al., 1990;

Ling and Zielinski, 1993; Libich and Harauz, 2002)showed that the truncated protein of OsDEX1 (aminoacids 336 to 634), containing 3 EF-hands motifs, wasshifted in an acrylamide-SDS gel after incubation with1 mM CaCl2. However, when Ca2+ was chelated by

adding EGTA, no shift in fragment migration wasdetected. In addition, when an isoform of OsDEX1 thefirst, third, fourth, fifth, and sixth residues of the con-served calcium binding domain were muted was in-cubated with CaCl2, no shift was observed (Fig. 8C).Importantly, when the mutated OsDEX1 cDNA drivenby DEX1 promoter (1.7 Kb) was introduced into theArabidopsis dex1 mutant, the transgenic plants homo-zygous for dex1 failed to produce viable pollen grains(Fig. 8, D to G). These results identify OsDEX1 as a Ca2+

binding protein and provide evidence for a pivotal roleof Ca2+ binding in the function of OsDEX1.

OsDEX1 Is Required for Adequate Ca2+ Homeostasisin Tapetum

The abnormal tapetal cell death phenotype and thealteration of the Ca2+ binding ability of a truncatedOsDEX1 protein suggested that OsDEX1 might regu-late tapetal cell death by affecting Ca2+ distribution inthe tapetal cells, as suggested by previous reports(Wyllie, 1980; Cohen and Duke, 1984). To test this,yellow cameleons 3.6 (YC3.6) expressed under thecontrol of the constitutive UBI10 promoter, a powerfultool monitoring the spatio-temporal dynamics of Ca2+

fluxes in different tissues of Arabidopsis and rice (Krebset al., 2012; Nehera et al., 2015), was used to monitorCa2+ homeostasis in rice tapetum. There are three de-tectable somatic cell layers in the rice anther at stage 9,i.e. the epidermis, endothecium, and tapetum. In Figure9, the anther images show the morphology of tapetalcells, which is consistent with a previous report (Zhaoet al., 2015). PM-YC3.6, which is a plasma membranetargeted version of YC3.6, is able to distinguish thefaint Ca2+ signal at the cytosolic side of the plasmamembrane from the strong autofluorescence of anthers.

Figure 8. Recombinant OsDEX1 has Ca2+ binding activity. A, 3Dstructure of EF-hand motif of YesW. B, 3D structure of EF-hand motif ofOsDEX1. C, In vitro Ca2+ binding assay shows that OsDEX1 has Ca2+

binding activity. D to G, Failure in complementation by OsDEX1 withthe mutated Ca2+ binding sites in dex1. Insets show pollen tested byAlexander staining using bright field microscopy. Bar = 10 mm.

Figure 7. A to D, Transgenic lines containing Pro:DEX1:OsDEX1 in theArabidopsis dex1 mutant display rescued male fertility. Insets show thepollen tested by Alexander staining using bright field microscopy. Ws,wild-type plant of Wassileskija ecotype. Bar = 10 mm.





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During anther development from stage 8 to stage 10,only a faint Ca2+ signal was observed in the wild-typeanthers. In sharp contrast, in the osdex1 mutant, theplasma membrane-localized Ca2+ reporter indicated adramatic increase of intracellular Ca2+ concentration.in the tapetal cells specifically initiating from stage 9,that was even further enhanced at stage 10 (Fig. 9, A toH). These results identify rapid and strong elevations ofintracellular Ca2+ concentration that occur stage specificonly in osdex1 and the coincide with the establishmentof tapetal phenotypes in this mutant. These findingssupport the conclusion that the increase in [Ca2+] spe-cifically observed in osdex1 represents an importantfacet of the cell death ongoing in these cells and there by

attribute a crucial function for maintaining appropriatecellular [Ca2+] homeostasis to OsDEX1.

In another assay for cytosolic [Ca2+] homeostasis,cytoplasmic NES-YC3.6 or plasma membrane targetedPM-YC3.6 were expressed alone or coexpressed to-gether with either OsDEX1 protein or non-Ca2+ bindingmOsDEX1 protein transiently in tobacco epidermalcells. Consistent with the results in rice tapetal cells,[Ca2+]PM was lower when PM-YC3.6 was coexpressedwith OsDEX1 compared to cell that did only expressPM-YC3.6 while [Ca2+]cyt was elevated when NES-YC3.6was coexpressedwithOsDEX1 compared to cells that onlyexpressed NES-YC3.6. Importantly, the Ca2+ concen-tration was not affected when PM-YC3.6 or NES-YC3.6

Figure 9. The Ca2+ concentration calculated by emission ratios between YFP and CFP intensities using confocal laser scanningmicroscope in rice anthers and tobacco epidermal cells. A to H, Ca2+ concentration on plasma membrane in tapetal cells of wildtype (A–D) and osdex1 (E and F) from stage 8 to stage 11. I to T, Comparison of cytosolic (I to N) and plasmamembrane (O–T) Ca2+

concentration in tobacco epidermal cells overexpressed with OsDEX1 (J and P) or mOsDEX1 (M and S). I, L, O, and R, Control ofYC3.6 overexpression. K, N, Q, and T, Statistical analysis of ratios between YFPand CFP intensities in the cells (K and N) and onthe plasma membrane (Q and T).


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were coexpressed with mOsDEX1, which lacks Ca2+

binding activity (Fig. 9). These results reveal that theexpression of OsDEX1 modulates the Ca2+ homeostasisin the cells, which is considered as a key mechanism forcell death regulation (Giorgi et al., 2008).To determine the subcellular localization of OsDEX1

at which this protein impacts on cellular [Ca2+] dynam-ics, the full-length OsDEX1 coding region was fusedwith theGFP at its C terminus, and transiently expressedin tobacco epidermal cells. The OsDEX1-GFP signal wasdetected on the ER (Supplemental Fig. S10), which is inagreement with previous reports suggesting that theER is a main storage site for Ca2+ in the cell (Pozzanet al., 1994). Considering the results described above,we therefore hypothesize that OsDEX1 may regulatetapetal cell death via modulating Ca2+ homeostasis be-tween the ER and other cellular compartments.

DISCUSSION

Rice is one of the most important foods for the globalpopulation. Rice yield improvement is largely depen-dent on hybrid breeding that employs male sterile lines.Although much progress has been made toward un-derstanding the molecular mechanisms underlyingplant male development, the role of Ca2+ during antherdevelopment has remained elusive. In this study, wehave demonstrated that an FG-GAP domain-containingprotein, OsDEX1, is required for pollen wall develop-ment and tapetum function in a way has not been elu-cidated in previous descriptions of the Arabidopsis dex1mutant (Paxson-Sowders et al., 2001). The evolutionaryimportance of the OsDEX1-associated pathway is sup-ported by the fertility restoration of dex1 by OsDEX1.

OsDEX1 Affects Tapetal Function andAnther Development

The primexine functions as a template for sporopol-lenin deposition, which is critical for pollen development.

Several genes have been identified that influence pri-mexine formation in Arabidopsis (Paxson-Sowderset al., 2001; Ariizumi et al., 2004; Chang et al., 2012;Sun et al., 2013), while no gene has been identified inrice. In this study, we identified the ortholog of Arabi-dopsis DEX1 in rice, which shares a similar functionwith Arabidopsis DEX1 in regulating primexine for-mation and male fertility (Paxson-Sowders et al., 2001).Similar to dex1, osdex1 showed defects in plasmamembrane undulation as well as primexine formation,resulting the failure in exine formation (Fig. 3). There-fore, DEX1 appears to play an evolutionarily conservedrole in male reproduction in model dicot Arabidopsisand monocot rice plants.

Primexine is a structure located at the periphery ofthe haploid microspores; however, it is believed thatprimexine formation is controlled by sporophytic cells.Evidence for this comes from reported primexine de-fective mutants, such as dex1, nef, hkm, and rpg1, whoseheterozygous mutants showed normal primexine for-mation (Paxson-Sowders et al., 2001; Ariizumi et al.,2004; Chang et al., 2012; Sun et al., 2013). Tapetal celldeath is of vital importance for exine formation(Sorensen et al.,2003; Li et al., 2006; Zhang et al., 2008;Aya et al., 2009; Xu et al., 2010; Niu et al., 2013; Jiet al.,2013). Although its role in primexine formationhas not been described previously, we agree thattapetal cell death is also required for primexine for-mation. In a previous report of Arabidopsis DEX1(Paxson-Sowers et al., 2001), mutant defects in micro-spore release from the tetrad, tapetal function as well asa biochemical characterization of DEX1 have not beenreported. In this study, we show that the newly pro-duced microspores within the osdex1 tetrad are coveredby an electron-dense matrix, and that microspore re-lease into the lobe is inhibited until at least stage 9.Immunolabeling and aniline blue assays indicate thatdegradation of microspore callose is abnormal in osdex1(Figs. 5 and S5), which has not been reported in Ara-bidopsis mutants such as dex1, npg1, hkm, rpg1 rpg2,npu, and nef (Paxson-Sowders et al., 2001; Ariizumi

Figure 10. Model for OsDEX1 in anther develop-ment. Transcription factors such as TIP2, TDR, PTC1,and EAT1 function as master valves to switch celldeath signaling on or off; OsDEX1 buffers the Ca2+

concentration in the cells to function as a componentof cell death signaling.







et al., 2004; Ariizumi et al., 2005; Guan et al., 2008;Chang et al., 2012, Sun et al., 2013). In addition, diffusehalos that surround osdex1microspores at stage 10 onlycontain very low levels of callose, suggesting that someother polymers are aberrantly deposited onto the osdex1microspores. Taken together, the weaker staining ofcallose by callose antibody and aniline blue at stage 8b,the less compact matrix detected by TEM observationaround osdex1 tetrads, and the persistence of calloseuntil stages 9 and 10 suggest that callose degradationmay initiate early in osdex1, but then fails to completedue to defects in tapetal development.

As a nutritive tissue, tapetal function such as propercell death and the biosynthesis of sporollenin precur-sors is essential for pollen wall formation. Either pre-mature or delayed tapetal degradation frequentlycauses reduced male sterility (Balk and Leaver, 2001;Ku et al., 2003; Lee et al., 2004; Jung et al., 2005; Luoet al., 2006; Kawanabe et al., 2006; Li et al., 2006; Oshinoet al., 2007; Li et al., 2011; Aya et al.,2009; Tan et al.,2012; Niu et al.,2013; Ji et al., 2013; Fu et al., 2014; Koet al., 2014; Yang et al., 2014; Zhang et al., 2014). Weobserved a persistent and partially expanded tapetallayer in osdex1 until stage 11. However, compared towild-type tapetal cells that are rich in organelles at stage 9,osdex1 tapetal cells form abnormal vacuoles. Thevacuoles expand and encapsulate organelles, leading todegradation of the inclusions in the tapetal cells. Theseobservations suggest that expanding vacuoles maycontribute to an alternative form of tapetal cell death inosdex1, distinct from normal tapetal cell death (Li et al.,2006; Niu et al., 2013). Consistent with this, the tapetalcell death executorsOsAP25,OsAP37, andOsCP1 fail tobe induced in osdex1 (Supplemental Fig. S4), suggestingthat the normal protein degradation pathway involvedin the tapetal cell death is not activated in osdex1. Fur-thermore, compared with the cell death-deficient mu-tant tdr whose tapetum expansion is uniform (Li et al.,2006), the expansion of different tapetal cells in osdex1varies (Fig. 3), suggesting a different molecular mech-anism of OsDEX1 on regulating tapetal cell deathcompared with TDR.

Tapetal cell death is characterized by changes such asDNA fragmentation, protease activation, and cellshrinkage (Li et al., 2006; Aya et al., 2009; Xu et al., 2010;Phan et al., 2011; Li et al., 2011a; Niu et al., 2013; Fuet al., 2014; Fig. 10). Notably, DNA fragmentation isobserved from stage 8b, while tapetal cell shrinkageand protease induction are observed from stage 9 in thewild-type anthers. The link between DNA fragmenta-tion and cell death remains to be elucidated. DNAfragmentation is observed in the osdex1mutant (Fig. 4),but no cell shrinkage or protease induction could beobserved, suggesting that OsDEX1 may act as a com-ponent required for the tapetal cell death signal trans-duction (Fig. 10). The early appearance of DNAfragmentation in osdex1 could be explained by the ear-lier induction of endonuclease genes (SupplementalFig. S4). HowOsDEX1 regulates the expression of thesegenes requires further investigation.

OsDEX1 Is a Ca2+ Binding Protein

Ca2+ concentration in the cytoplasm, organelles, andcell wall is maintained within a certain range (Knight,2000; White, 2000). Ca2+ binding proteins are requiredfor the decoding of transient [Ca2+]cyt (Enslen et al.,1995; Snedden and Fromm, 1998; Day et al., 2002; Kudlaet al., 2010; Steinhorst and Kudla, 2013), and usuallycontain EF-hand motifs that are rich in negative aminoacid for the coordination with Ca2+. EF-hand usuallyform a helix-loop-helix structure (Day et al., 2002;Derbyshire et al., 2007; Rigden et al., 2011). However,there are some b-propeller structure proteins shown topossess Ca2+ binding activity (Cioci et al., 2006). It hasbeen reported that an eight-bladed b-propeller struc-ture protein, RG lyase YesW from saprophytic Bacillussubtilis, had the ability for Ca2+ binding, despite lackingthe canonical EF-hand motif (Ochiai et al., 2007). 3D-structure predictions show that OsDEX1 harbors ab-sheet-rich region linked by loops of negatively aminoacids that form a pocket for Ca atom, similar to YesW(Supplemental Figs. S9 and 8). The in vitro Ca2+ bind-ing assay indicates that truncated OsDEX1 has the Ca2+

binding activity, and the genetic complementation us-ing themutated OsDEX1 suggests that the Ca2+ bindingactivity is required for pollen development in vivo (Fig.8). Furthermore, OsDEX1 is an ER-localized protein(Supplemental Fig. S10), and osdex1 loss-of-functionmutant accumulated Ca2+ on the plasma membrane,while overexpressing OsDEX1 prevented the Ca2+ ac-cumulation on the plasma membrane (Fig. 9). Theseresults suggest a role for OsDEX1 in affecting the levelof Ca2+ on the plasma membrane (Fig. 10).

OsDEX1 Has a Conserved Function during PlantMale Reproduction

The pollen wall is essential for pollen grains resistant tovarious biotic and abiotic stresses in flowering plants. Inthis study, we identified and analyzed 25 DEX1-like pro-teins from flowering plants, lower plants, lower animals,fungi, and bacteria. Their phylogenetic relationship sug-gests that these genes probably share a common ancestorand have a conserved function. Notably, the proteins inflowering plants share one or two FG-GAP domains thatare required for the Ca2+ binding (Fig. 6). The proteinstructure similarity in flowering plants highlights the con-served and essential role of OsDEX1 and its homologs inpollen wall formation in flowering plants. To our knowl-edge, OsDEX1 is the first member of the DEX1 family thathas been identified as having Ca2+ binding activity.

In summary, we have demonstrated that the FG-GAPdomain protein OsDEX1 is able to bind Ca2+ to modu-late cellular Ca2+ homeostasis, and regulates tapetaldegradation and pollen wall formation. A conservedrole for DEX1-like proteins was established by the ge-netic complementation of Arabidopsis dex1 by OsDEX1.This finding provides important insights into the role ofCa2+in male reproduction in plants.


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MATERIALS AND METHODS

Plant Materials, Growth Conditions, and MolecularCloning of OsDEX1

Rice (Oryza sativa ssp. japonica, 9522) plants were grown in the paddy field ofShanghai JiaoTong University. Male sterile plants in the F2 progenies generatedby a cross between wild-type GuangLuAi species (indica) and the osdex1mutant(japonica) were selected for mapping. Twenty-four pairs of InDel molecularmarkers were designed based on the polymorphism between japonica and indicafor mapping. Further fine-mapping of OsDEX1was performed using the pre-viously published method from Li et al. (2006).

Characterization of Mutant Plant Phenotypes

Anthers from different developmental stages, as defined in Zhang et al.(2011), were collected based on the comparison and analysis of wild-type andosdex1 plants on glume length and morphology. Observation of anther de-velopment by semithin section analysis, TEM, and callose staining wereperformed according to previous studies (Fu et al., 2014; Aditya et al., 2015).For callose immunolabeling, sections were first deparaffinated using xylenebefore labeling with anticallose antibody (Meikle et al., 1991). Images werecaptured on a Zeiss A1 AxioImager using a black and white camera andZEN2012 software. Identical exposure times were used to enable compari-sons between stages and samples. Green immunostaining (Alexafluor-488)was viewed using Zeiss Filter Set 38; blue counterstaining (0.01% calcofluorwhite) was viewed using Zeiss filter set 49, and red staining (auto-fluorescence) was viewed using Zeiss filter set 43.

TUNEL Assay

Samples from wild-type and osdex1 anthers at different developmentalstages were collected. The TUNEL assay was performed as reported in Fuet al. (2014). The samples were analyzed under a fluorescence confocalscanner microscope (Leica SP5II system). Images were recorded using a HCXPL APO CS 20*0.7 DRY objective. The imaging parameters were as follows:image dimension (1024*1024); pinhole (2.19 airy unit); scanning speed(100 Hz); line average (3). The fluorescein signal was excited using the 488 nmlaser line of the Argon laser (30%). The image parameters for this channelwere as follows: 488 nm 48%; smart gain (817.0); offset (22.5%). The propi-dium iodide signal was excited using the 543 nm laser line of the HeNe 543.The image parameters for this channel were as follows: 543 nm 33%; smartgain (654.0); offset (22.5%). The overlays of fluorescein signal and propidiumiodide signal were shown as the TUNEL-positive signal. All pictures weretaken in the same setting.

Cloning of OsDEX1 and Plant Transformation

A 2.5 kb cDNA sequence of OsDEX1was amplified from the cDNA reversetranscripted by the RNA extracted from anthers of 9522. A 1.7 kb upstreamsequence of Arabidopsis DEX1 was amplified from the genomic DNA ofCol-0. The cDNA of mutated EF-hand motif 1 was amplified by the primersmEF1 F1 and mEF1 R1, and mEF1 F2 and mEF1 R2. The PCR products of theamplification were taken as the template for a second PCR, with the primersmEF1 F1 andmEF1 R2. The cDNA of mutated EF-hand motif 2 was amplifiedby the primers mEF2 F1 and mEF2 R1, and mEF2 F2 and mEF2 R2. The PCRproducts of the amplification were taken as the template for a second PCR,with the primers mEF2 F1 and mEF2 R2. The two fragments were ligatedtogether by NcoI and XbaI, which existed in the primer and the pBlueScriptSK, and then ligated with the 59 end of the cDNA to a full-length cDNA.Taking the full-length wild-type and mutated full-length cDNA as the tem-plates, the amplified fragments were cloned into the binary vector pCAM-BIA1301. Plasmids were then transferred into Agrobacterium tumefaciensGV3101 and Arabidopsis heterozygous dex1 plants. The transformants werescreened for the presence of transgene on hygromycin medium. Over100 positive transgenic plants in each transformation were obtained andgenotyped for the homozygous dex1 mutant background (primers used arelisted in Supplemental Table S1). The PM-YC3.6 (Krebs et al., 2012) weretransferred into A. tumefaciens EHA105. Calli induced from young panicles ofthe wild type 9522 and osdex1 mutant were used for transformation (Li et al.,2006). For transgenic plants, at least three independent lines were obtainedfor each construct.

qRT-PCR and In Situ Hybridization

Total RNA from corresponding tissues was isolated using TRI reagent fromrice tissues. Ninety milligrams of RNA was used to synthesis cDNA in eachsample using the Primescript RT reagent kit with genomicDNAeraser (Takara).qRT-PCR was performed with the lightCycler system (Roche) using SYBRPremix Ex Taq GC (Takara) according to the manufacturer’s instructions.Amplification was conducted following this protocol: 95°C for 2 min; 40 cyclesof 95°C for 5 s; and 55°C for 30 s; 72°C for 30 s. ACTIN (Supplemental Table S1)was used as an internal control, and a relative quantization method (D cyclethreshold) was used to quantify the relative expression level of target genes.Three biological replicates with three technique replicates eachwere included inproducing the statistical analysis and the error range analysis. In situ hybridi-zation was performed as described in Fu et al. (2014). Two OsDEX1 cDNAfragments generated by PCR were used for preparing antisense and senseprobes.

Phylogenetic Analysis

Multiple alignments were performed using Muscal 3.6 (http://www.ebi.ac.uk/Tools/msa/muscle/). A phylogenetic tree was constructed with thealigned sequences from the full-length region of OsDEX1. MEGA (version6.0; http://www.megasoftware.net/index.html) and the Neighbor-Jointingmethods were used with p-distance model and pairwise deletion and boot-strap (1000 replicates; random seed). The Max Parsimony method of MEGAwas also employed to support the Neighbor-Jointing tree, using the defaultparameter.

Heterologous Expression and In Vitro Ca2+ Binding Assay

Wild-type and EF-hands-mutated OsDEX1 cDNAwas amplified by primerscDNAFand cDNAR fusedwithGST, expressed inBL21DE3Escherichia coli cells.Proteins were purified using amylose andGSSH resin, respectively. The proteinextraction was incubated with 1 mMCaCl2 or 1 mMCaCl2, together with 1 mMEGTA at 4°C overnight. The mobility was detected in 10% SDS-PAGE.

Calcium Imaging

FRET-based Ca2+ imaging was performed as described in Krebs et al. (2012).Anthers at different stages from YC3.6 transgenic lines in wild type or osdex1were used for analyses. The samples were analyzed using a fluorescence con-focal scanner microscope (Leica SP5II system). The objective, the imaging di-mension, and scanning speed were same as above. The pinhole was 2.32 airyunit. The CFP signal was excited using the 458 nm laser line of the Argon laser(30%). The image parameters for the YFP channel were as follows: smart gain(1028.1); offset (216.1%). The image parameters for this channel were as fol-lows: smart gain (763.0); offset (212.7%). The ratios between CFP and YFPsignal intensity, subtracting the background fluorescence in a region outside theanthers, were calculated. For the ratio calculation, the parameters for imagescaling were as follows: Min (0); Max (2).

For the Ca2+ imaging in tobacco cells, the epidermis cells transformed withcorrespondingplasmids for 2 dwere observed. The objective, imaging dimension,and scanning speedswere same as above. The pinholewas 1.00 airy unit. TheCFPsignal was excited using the 458 nm laser line of the Argon laser (50%). The imageparameters for this channelwere as follows: 458 nm33%; smart gain (761.0); offset(0.1%). The image parameters for YFP channelwere as follows: smart gain (531.0);offset (21.3%). For the statistical analysis of [Ca2+]cyt, the ratio of fluorescenceintensity in channel CFP and YFP in the whole region of the cell was measured andcalculatedas described (Behera et al., 2015). For the statistical analysis of [Ca2+]PM, theratios of fluorescence intensity in channels CFP and YFP around the plasmamembrane were measured (n . 15 each).

Accession Numbers

Sequence data from this article can be found in the GenBank/EMBLdata libraries under accession numbers XP_015631235; XP_006651977;XP_008666953; XP_003563417; XP_004981208; XP_002466172; ERN09669;NP_566343; CDY67523; XP_008388127; XP_006573337; XP_006576567;XP_003604604; XP_001782562; XP_002973603; XP_001703106; CCW71639;XP_780158; XP_005093156; EKC30460; XP_009052863; WP_012257615;WP_022229450; NP_070770.






http://www.ebi.ac.uk/Tools/msa/muscle/

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http://www.megasoftware.net/index.html


Supplemental Data

The following supplemental materials are available.

Supplemental Figure S1. The model of anther development from stage 5 tostage 9.

Supplemental Figure S2. Transmission electron micrographs of the an-thers from the wild type (A) and the osdex1 mutant (B) at stage 8a.Bars = 20 mm.

Supplemental Figure S3. Expression of genes associated with sporopol-lenin synthesis and transport genes in the wild type and osdex1 duringpollen development. Bars = SE.

Supplemental Figure S4. Protease genes and endonuclease genes expres-sion in osdex1 and tapetum cell death deficient mutants.

Supplemental Figure S5. Callose degradation is abnormal in osdex1.

Supplemental Figure S6. Molecular identification and expression patternof OsDEX1.

Supplemental Figure S7. Phenotype of osdex1-2 and osdex1-4. A, Wild-typeflower (left) and osdex1-2 mutant flower (right) before anthesis.

Supplemental Figure S8. Alignment of OsDEX1 and DEX1.

Supplemental Figure S9. Structure prediction of OsDEX1 and DEX1.

Supplemental Figure S10. Sublocalization analysis using confocal laserscanning microscope of OsDEX1 in tobacco epidermal cell.

Supplemental Table S1. Primers used in this study.

Supplemental Data Set 1. Protein sequences used for phylogenetic tree.

ACKNOWLEDGMENTS

We thank Lu Zhu, Jie Xu, and Wanwan Zhu for TEM observation at theInstrumental Analysis Center of Shanghai Jiao Tong University. We also thankLisa O’Donovan from the University of Adelaide for assistance with calloseimmunolabeling.

Received August 16, 2016; accepted September 19, 2016; published September23, 2016.

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